Method for the diagnosis of sepsis with determination of CA 19-9

ABSTRACT

Method for early differential diagnosis and detection, prognosis and assessment of the severity and therapy-accompanying assessment of the course of sepsis and sepsis-like systemic infections, in which, preferably with determination of at least one further parameter suitable for sepsis diagnosis, the amount of CA 19-9 in a biological fluid of a patient who is suffering from sepsis or is suspected of having sepsis is determined and conclusions are drawn from the determined amount of CA 19-9 with regard to the presence, the expected course, the severity and/or the success of initiated measures for the treatment of the sepsis.

The present application is a nationalization of PCT Application SerialNo. PCT/EP02/13392, filed Nov. 27, 2002, which claims priority toEuropean application No. 01128850.3, filed Dec. 4, 2001.

The invention relates to a novel method for sepsis diagnosis, in which,or in the course of which, the parameter CA 19-9 known per se as atypical tumour marker in medical diagnosis is determined.

The invention is based on the detection for the first time of greatlyincreased concentrations of CA 19-9 in sera of patients who have beendiagnosed as having a sepsis on the basis of clinical findings withsimultaneously increased serum concentrations of the known sepsis markerprocalcitonin.

The present invention has its origin in intensive research work by theApplicant in relation to further improvements of the diagnosis andtherapy of inflammations of infectious aetiology and sepsis.

Inflammations are defined very generally as certain physiologicalreactions of an organism to different types of external effects, suchas, for example, injuries, burns, allergens, infections bymicroorganisms, such as bacteria and fungi and viruses, to foreigntissues which trigger rejection reactions, or to certain inflammatoryendogenous conditions of the body, for example in autoimmune diseasesand cancer. Inflammation may occur as harmless, localized reactions ofthe body but also typical features of numerous serious chronic and acutediseases of individual tissues, organs, organ parts and tissue parts.

In sepsis or septic shock, inflammation-specific reaction cascadesspread in an uncontrolled manner over the whole body and may becomelife-threatening in the context of an excessive immune response.Regarding the current knowledge about the occurrence and the possiblerole of individual groups of endogenous sepsis-specific substances,reference is made, for example, to A. Beishuizen et al., “EndogenousMediators in Sepsis and Septic Shock”, Advances in Clinical Chemistry,Vol. 33, 1999, 55-131; and C. Gabay et al., “Acute Phase Proteins andOther Systemic Responses to Inflammation”, The New England Journal ofMedicine, Vol. 340, No. 6, 1999, 448-454. Since the understanding ofsepsis and related systemic inflammatory diseases, and hence also therecognized definitions, have changed in recent years, reference is alsomade to K. Reinhart et al., “Sepsis and septischer Schock” [Sepsis andseptic shock], in: Intensivmedizin, Georg Thieme Verlag, Stuttgart, NewYork, 2001, 756-760, where a modern definition of sepsis is given. Inthe context of the present invention, the term sepsis used is based onthe definitions given in the stated references.

Whereas at least in Europe the systemic bacterial infection detectableby a positive blood culture long characterized the term sepsis, sepsisis now primarily understood as being systemic inflammation which iscaused by infection. Said transformation in the understanding of sepsishas resulted in changes in the diagnostic approaches. Thus, the directdetection of bacterial pathogens was replaced or supplemented by complexmonitoring of physiological parameters and, more recently, in particularby the detection of certain endogenous substances involved in the sepsisprocess or in the inflammatory process, i.e. specific “biomarkers”.

Of the large number of mediators and acute phase proteins which areknown to be, or presumed to be, involved in an inflammatory process, theones which are suitable for purposes of clinical sepsis diagnosis are inparticular those which occur with high sensitivity and specificity insepsis or certain phases of a sepsis or whose concentrations change in adramatic and diagnostically significant manner and which moreover havethe stabilities required for routine determinations and reach highconcentration values. For diagnostic purposes, the reliable correlationof pathological process with the respective biomarker is of primaryimportance, without there being any need to know exactly its role in thecomplex cascade of the endogenous substances involved in the sepsisprocess.

A known endogenous substance particularly suitable as a sepsis biomarkeris procalcitonin. Procalcitonin is a prohormone whose serumconcentrations reach very high values under the conditions of a systemicinflammation of infectious aetiology, whereas it is virtuallyundetectable in healthy persons. High values of procalcitonin are alsoreached in a relatively early stage of a sepsis so that thedetermination of procalcitonin is also suitable for early diagnosis of asepsis and for early distinguishing of a sepsis caused by infection fromsevere inflammations which have other causes. The procalcitonindetermination is furthermore particularly valuable fortherapy-accompanying observation of the course of a sepsis. Thedetermination of procalcitonin as a sepsis marker is the subject of thepublication by M. Assicot et al., “High serum procalcitoninconcentrations in patients with sepsis and infection”, The Lancet, Vol.341, No. 8844, 1993, 515-518; and the patents DE 42 27 454 C2 and EP0656 121 B1 and U.S. Pat. No. 5,639,617. Reference is hereby madeexpressly to said patents and to early literature references mentionedin said publication for supplementing the present description. In recentyears, the number of publications on the subject of procalcitonin hasgreatly increased. Reference is therefore also made to W. Karzai et al.,“Procalcitonin—A New Indicator of the Systemic Response to SevereInfection”, Infection, Vol. 35, 1997, 329-334; and M. Oczenski et al.,“Procalcitonin: a new parameter for the diagnosis of bacterial infectionin the peri-operative period”, European Journal of Anaesthesiology 1998,15, 202-209; and furthermore H. Redl et al., “Procalcitonin releasepatterns in a baboon model of trauma and sepsis: Relationship tocytokines and neopterin”, Crit Care Med 2000, Vol. 28, No. 11,3659-3663; and H. Redl et al., “Non-Human Primate Models of Sepsis”, in:Sepsis 1998; 2:243-253; and the further literature references citedtherein, as typical of recent published reviews.

The availability of the sepsis marker procalcitonin has givenconsiderable impetus to sepsis research, and intensive efforts are nowbeing made by the Applicant to find further biomarkers which cansupplement the procalcitonin determination and/or are capable ofproviding additional information for purposes of fine diagnosis ordifferential diagnosis. Thus, a search is being made in particular forfurther biomarkers for sepsis diagnosis, whose serum or plasma levelsare regularly increased but whose determination does not simplyduplicate the results of the procalcitonin determination but providesadditional information, in particular on the stage of the sepsisprocess, i.e. information which can rather be assigned to the progressof the sepsis, and/or on the initial or principal organ of a septicprocess, i.e. localizing information. The aim is in the end theselection of a set of sepsis parameters which are simultaneouslydetermined in the case of sepsis patients or potential sepsis patients,for example using the so-called chip technology or immunochromatographicmethods (“point of care” or POC determinations), and in their totalityprovide an information pattern which clearly surpasses the informationvalue of the determination of only one individual parameter.

The search for potential novel sepsis biomarkers is, however,complicated by the fact that frequently very little or nothing is knownabout the exact function or about the exact reasons for the occurrenceof certain endogenous substances which are involved in the inflammatoryor septic process.

Since the endogenous substances increased during sepsis are part of thecomplex reaction cascade of the body, not only are such substances alsoof diagnostic interest but attempts are currently also being made, withconsiderable effort, to intervene therapeutically in the sepsis processby influencing the formation and/or the concentration of individualsubstances of this type, in order to stop as early as possible thesystemic spread of the inflammation, which spread is observed, forexample, during sepsis. In this context, endogenous substances whichhave been shown to be involved in the sepsis process are also to beregarded as potential therapeutic targets.

The results of the experimental testing of a fruitful purelyhypothetical approach to the determination of further potential sepsismarkers are to be found in DE 198 47 690 A1 and WO 00/22439. There, itis shown that, in the case of sepsis, not only is the concentration ofthe prohormone calcitonin significantly increased but also significantlyincreased concentrations can be observed for other substances which maybe included among the peptide prohormones. In particular, the peptideprohormones pro-enkephalin, pro-gastrin-releasing peptide (proGRP),pro-endothelin-1, pro-brain-natriuretic-peptide (pro-BNP),pro-atrial-natriuretic-peptide (pro-ANP), pro-leptin,pro-neuropeptide-Y, pro-somatostatin, pro-neuropeptide-YY,pro-interleukin-6 or pro-interleukin-10 may be mentioned in thiscontext. While the phenomenon described is well documented, the causesof the increase in the concentrations of prohormones during sepsis arestill substantially unexplained.

In the present Application, a result of another hypothetical approach inthe search for further biomolecules suitable for sepsis diagnosis is nowreported. It is based on the results of measurements of thephysiological concentrations of biomarkers, which have been regarded todate as typical tumour markers and have therefore been clinicallydetermined substantially for purposes of tumour diagnosis, in biologicalsamples, in particular serum samples, of sepsis patients for whom noclinical findings at all indicate the presence of tumours.

Surprisingly, it has been found that some biomolecules regarded to dateas typical tumour markers are also significantly increased in sepsis.This indicates that these are not formed in a tumour-specific manner butrather indicate a critical physiological process which also affectstissues or organs which release these tumour markers. Although all theconcentrations of the. biomolecules in question are increased duringsepsis with high sensitivity, as shown in this Application andsimultaneously filed further Applications, there is at the same time nocorrelation of the measured values with the likewise significantlyincreased procalcitonin concentrations, i.e. both parameters are foundto be increased in individual patients but in some cases to verydifferent extents.

The present invention is based on the evidence, obtained for the firsttime, that significantly increased physiological concentrations of CA19-9 are found in the case of bacterial sepsis, which makes theseparameters, particularly in combination with the determination offurther sepsis parameters, suitable for differential sepsis diagnosis.

The method according to the invention and certain preferred embodimentsthereof are defined in more detail in Claims 1 to 6.

It was not known to date that the measurable concentrations of CA 19-9,in particular serum concentrations, are significantly increased in thecase of sepsis and that a determination of the concentration of CA 19-9may therefore also be important for sepsis diagnosis.

On the basis of the present invention, it is possible to use thedetermination of CA 19-9 also in the course of a diagnostic sepsis testmethod. Of particular interest is the suitability of CA 19-9 as aprognosis marker and marker for monitoring the course of sepsis, inparticular as part of a combination measurement with other markers.

In addition to a combination with a procalcitonin measurement, acombination of the measurement of CA 19-9 with the determination ofother markers for sepsis and systemic inflammations, which have beenregarded to date as typical tumour markers, is particularly suitable,especially with CA 125, S100B or S100A proteins involved in theregulation of inflammations, or with the determination of the novelsepsis markers inflammin (DE 101 19 804.3) and CHP (DE 101 31 922.3)described in the below-mentioned prior unpublished German PatentApplications of the Applicant, and/or with the determination of solublecytokeratin fragments, in particular of the recently found solublecytokeratin-1 fragments (sCY1F; DE 101 30 985.6) and of the known tumourmarkers CYFRA-21 or TPS, and/or with the determination of one or more ofthe above-mentioned prohormones. A simultaneous determination of theknown inflammation parameter C-reactive protein (CRP) can also beprovided. On the basis of the novel results described in thisApplication and the parallel Applications, a combination withmeasurements of known biomolecules or biomolecules still to be found,which are suitable as tissue- or organ-specific inflammation markers,should also be considered generally for fine sepsis diagnosis.

The content of said prior Applications of the Applicant is to beregarded as part of the disclosure of the present Application by expressreference to these Applications.

CA 19-9 (cancer antigen 19-9) or GICA (gastrointestinal cancer antigen)is defined as a glycolipid which has a molar mass of about 36 kD and isidentifiable by reaction with a certain specific monoclonal antibody(1116 NS 19-9; cf. Koprowski et al., Somatic Cell Genet 1979,5:957-972). Its biological function is unknown.

Owing to its high specificity, it plays an important role in thediagnosis, therapeutic monitoring and monitoring of the course ofgastrointestinal carcinomas, in particular in the case of pancreaticcarcinoma, hepatobiliary carcinoma (carcinoma of the liver, carcinoma ofthe bile ducts) and carcinoma of the stomach (cf. Lothar Thomas(editor): Labor und Diagnose, Section 34.3, pages 966-969, 5th Edition,1998, TH-Books Verlagsgesellschaft).

It is known that increased levels of CA 19-9 can also be measured inorders of magnitude of about 20% of cases also in some patients withnonmalignant inflammatory diseases, such as cholecystitis andobstructive icterus, cholelithiasis, cholecystolithiasis, acutechlolangitis, toxic hepatitis and other liver diseases (cf. LotharThomas, loc cit; M. J. Duffy, Ann Clin Biochem 1998; 35; 364-370).

According to our knowledge, no systematic CA 19-9 measurements have asyet been carried out in the case of patients suffering from sepsis, andnothing was known up to now concerning significantly increased measuredvalues of the typical tumour markers CA 19-9 in the vast majority ofcases in patients with systemic inflammations (sepsis).

A substantial increase in the CA 19-9 concentrations in the predominantnumber of sepsis patients was found for the first time in thedeterminations which are described in the following experimental reportwith reference to two figures.

In the figures:

FIG. 1 shows the results of the determination of CA 19-9 in the sera of171 sepsis patients in comparison with a group of 50 control persons(blood donors); and

FIG. 2 shows the correlation of the results of the CA 19-9determinationsof the 171 sepsis patients of FIG. 1 with the results of theprocalcitonin determination.

Experimental Report:

The concentrations of the tumour marker CA 19-9 were measured in 171sera of sepsis patients in whom high values of the sepsis markerprocalcitonin (PCT) had been found, using a commercial assay for thedetermination of CA 19-9 (KRYPTOR-CA19-9 from B.R.A.H.M.S DiagnosticaGmbH). In 62% of the sera, greatly increased CA 19-9 concentrations(more than 20 U/ml) were found.

A graph of the measured results is shown in FIG. 1.

If the CA 19-9 values measured for individual sera are compared with thevalues measured for PCT, no positive correlation is found in the sensethat the highest CA 19-9 values are also found in sera in which high PCTconcentrations were found. FIG. 2 shows the correlations found in thecase of such a comparison. It is evident that high CA 19-9 values (upperthird of the diagram) are obtained at moderate PCT concentrations, andmoderate values for CA 19-9 at very high PCT concentrations (right thirdof the diagram).

The fact that the results of the CA 19-9 determination are substantiallyindependent from those of the PCT determination shows that differenteffects are measured in spite of the increased values for bothparameters in the case of sepsis, which means that the measurement ofboth parameters provides more information than the measurement of onlyone of the parameters.

The combination of the determination of CA 19-9 with that of one or moresepsis markers is therefore suitable for improving the fine diagnosis ofsepsis and for improving the prognosis of the course of the disease andfor therapy-accompanying monitoring of the course in sepsis patients, itclearly being hoped that the interpretation of the results of suchcombined determinations based on the exact evaluation of individualcases documented as completely as possible (with, for example,information about the type of infection, reason for and course of thesepsis disease, characteristic data on the age and sex of the patients)will be steadily improved with the number of cases evaluated.

The determination of CA 19-9 can be carried out by any desired suitabledetection method, although the determination in a body fluid of apatient by an immunodiagnostic method using suitable selectiveantibodies appears to be the most advantageous from practical points ofview. Commercial assays for the determination of CA 19-9 are alreadyavailable and can also be used in the context of the present invention.Where necessary, good accuracy of measurement in the measuring rangerelevant for the sepsis diagnosis must be ensured.

Thus, the determination of CA 19-9 can be carried out for earlydifferential diagnosis and for detection and for the preparation of aprognosis, for assessment of the degree of severity and fortherapy-accompanying assessment of the course of sepsis, by determiningthe content of CA 19-9 in a sample of biological fluid of a patient insuch a method and drawing conclusions about the presence of a sepsisfrom the detected presence and/or amount of CA 19-9 and correlating theresult obtained with the severity, the progress of the sepsis and/or thetissue or organ most greatly affected by the sepsis and choosing thepossible treatments accordingly and/or estimating the prospects of thetreatments.

1. A method for assisting in the diagnosis of sepsis, said methodcomprising testing a biological fluid of a patient who, based onclinical findings, has sepsis-associated symptoms, for an elevatedamount of CA19-9 as compared to a control group, wherein an elevatedamount of CA19-9 in said sample is indicative of sepsis.
 2. The methodof claim 1, wherein the elevated amount of CA19-9 is determined by animmunodiagnostic assay method.
 3. The method of claim 1, wherein saidmethod is carried out in the course of a multiparameter determination inwhich at the same time at least one further sepsis parameter isdetermined.
 4. The method of claim 3, wherein said at least one furthersepsis parameter determined in the course of the multiparameterdetermination is selected from the group consisting of procalcitonin,CA125, S100B, S100A proteins, CYFRA 21, TPS, soluble cytokeratin-1fragments (sCY1F), inflammin, CHP, peptide prohormones and theC-reactive protein (CRP).
 5. The method of claim 3, wherein saidmultiparameter determination is carried out as a simultaneousdetermination using a measuring apparatus operating according to thechip technology or immunochromatographic method.
 6. The method accordingto claim 5, wherein said measuring apparatus provides a complex measuredresult and wherein the evaluation of said complex measured result iscarried out using a computer program.
 7. The method of claim 1, whereinsaid biological fluid is serum.
 8. The method of claim 1, wherein saidsepsis is a bacterial sepsis.
 9. The method of claim 3, wherein saidmethod is carried out in the course of a multiparameter determination inwhich at the same time other sepsis parameters are determined, and aresult in the form of a set of measured variables is obtained andevaluated for sepsis diagnosis.